Microrna-controlled recombinant vaccinia virus and use thereof

Abstract

It is intended to provide a vaccinia virus that specifically proliferates in a cancer cell and destroys the cancer cell and to provide use of the virus in cancer treatment. The present invention provides a microRNA-controlled vaccinia virus, in which a target sequence of a microRNA less expressed in a cancer cell than in a normal cell is inserted in a 3′ untranslated region of B5R gene associated with viral proliferation in a vaccinia virus, wherein the microRNA-controlled vaccinia virus specifically proliferates in the cancer cell and has an oncolytic property that destroys the cancer cell.

1 . A microRNA-controlled vaccinia virus, in which a target sequence of a microRNA less expressed in a cancer cell than in a normal cell is inserted in a 3′ untranslated region of B5R gene associated with viral proliferation in a vaccinia virus, wherein the microRNA-controlled vaccinia virus specifically proliferates in the cancer cell and has an oncolytic property that specifically destroys the cancer cell. 
     
     
         2 . The microRNA-controlled vaccinia virus according to  claim 1 , wherein the microRNA expressed in the normal cell represses the expression of the B5R gene to reduce the proliferative capacity of the microRNA-controlled vaccinia virus in the normal cell. 
     
     
         3 . The microRNA-controlled vaccinia virus according to  claim 1  or  2 , wherein the B5R gene into which the microRNA target sequence is inserted in its 3′ untranslated region is introduced into an attenuated vaccinia virus lacking a portion or the whole of its B5R gene. 
     
     
         4 . The microRNA-controlled vaccinia virus according to  claim 1  or  2 , wherein the vaccinia virus is an LC16 strain or an LC16mO strain. 
     
     
         5 . The microRNA-controlled vaccinia virus according to  claim 3 , wherein the vaccinia virus is an LC16m8 strain lacking a portion of its B5R gene or an m8Δ strain lacking the whole of its B5R gene. 
     
     
         6 . The microRNA-controlled vaccinia virus according to  claim 1 , wherein the microRNA less expressed in a cancer cell than in a normal cell is selected from the group consisting of let-7a (SEQ ID NO: 1), let-7b (SEQ ID NO: 2), let-7c (SEQ ID NO: 3), let-7d (SEQ ID NO: 4), let-7e (SEQ ID NO: 5), let-7f (SEQ ID NO: 6), miR-9 (SEQ ID NO: 7), miR-15a (SEQ ID NO: 8), miR-16-1 (SEQ ID NO: 9), miR-21 (SEQ ID NO: 10), miR-20a (SEQ ID NO: 11), miR-26a (SEQ ID NO: 12), miR-27b (SEQ ID NO: 13), miR-29a (SEQ ID NO: 14), miR-29b (SEQ ID NO: 15), miR-29c (SEQ ID NO: 16), miR-30a (SEQ ID NO: 17), miR-30d (SEQ ID NO: 18), miR-32 (SEQ ID NO: 19), miR-33a (SEQ ID NO: 20), miR-34a (SEQ ID NO: 21), miR-92a (SEQ ID NO: 22), miR-95 (SEQ ID NO: 23), miR-101 (SEQ ID NO: 24), miR-122 (SEQ ID NO: 25), miR-124 (SEQ ID NO: 26), miR-125a (SEQ ID NO: 27), miR-125b (SEQ ID NO: 28), miR-126 (SEQ ID NO: 29), miR-127 (SEQ ID NO: 30), miR-128 (SEQ ID NO: 31), miR-133b (SEQ ID NO: 32), miR-139-5p (SEQ ID NO: 33), miR-140 (SEQ ID NO: 34), miR-141 (SEQ ID NO: 35), miR-142 (SEQ ID NO: 36), miR-143 (SEQ ID NO: 37), miR-144 (SEQ ID NO: 38), miR-145 (SEQ ID NO: 39), miR-155 (SEQ ID NO: 40), miR-181a (SEQ ID NO: 41), miR-181b (SEQ ID NO: 42), miR-181c (SEQ ID NO: 43), miR-192 (SEQ ID NO: 44), miR-195 (SEQ ID NO: 45), miR-198 (SEQ ID NO: 46), miR-199a (SEQ ID NO: 47), miR-199b-5p (SEQ ID NO: 48), miR-200a (SEQ ID NO: 49), miR-203 (SEQ ID NO: 50), miR-204 (SEQ ID NO: 51), miR-205 (SEQ ID NO: 52), miR-217 (SEQ ID NO: 53), miR-218 (SEQ ID NO: 54), miR-219-5p (SEQ ID NO: 55), miR-220a (SEQ ID NO: 56), miR-220b (SEQ ID NO: 57), miR-220c (SEQ ID NO: 58), miR-222 (SEQ ID NO: 59), miR-223 (SEQ ID NO: 60), miR-224 (SEQ ID NO: 61), miR-345 (SEQ ID NO: 62), and miR-375 (SEQ ID NO: 63). 
     
     
         7 . The microRNA-controlled vaccinia virus according to  claim 1 , wherein the microRNA-controlled vaccinia virus is deficient in one or more gene(s) whose loss of function resulting from deletion of the gene(s) is compensated for in the cancer cell, but is not compensated for in the normal cell. 
     
     
         8 . The microRNA-controlled vaccinia virus according to  claim 7 , wherein the microRNA-controlled vaccinia virus is deficient at least in a thymidine kinase gene. 
     
     
         9 . The microRNA-controlled vaccinia virus according to  claim 8 , wherein the microRNA-controlled vaccinia virus is further deficient in a hemagglutinin (HA) gene. 
     
     
         10 . The microRNA-controlled vaccinia virus according to  claim 9 , wherein the microRNA-controlled vaccinia virus is further deficient in an F fragment. 
     
     
         11 . The microRNA-controlled vaccinia virus according to  claim 8 , wherein the microRNA-controlled vaccinia virus is further deficient in a VGF gene. 
     
     
         12 . A pharmaceutical composition for cancer treatment, comprising a microRNA-controlled vaccinia virus according to  claim 1 . 
     
     
         13 . A microRNA-controlled vaccinia virus vector comprising a foreign DNA introduced in a microRNA-controlled vaccinia virus according to  claim 1 . 
     
     
         14 . The microRNA-controlled vaccinia virus vector according to  claim 13 , wherein the foreign DNA is a marker DNA, a therapeutic gene having cytotoxic effect or immunostimulating effect, or a DNA encoding a cancer, viral, bacterial, or protozoal antigen. 
     
     
         15 . A pharmaceutical composition for cancer treatment or for use as a vaccine against a cancer, a virus, a bacterium, or a protozoan, comprising a microRNA-controlled vaccinia virus vector according to  claim 13 . 
     
     
         16 . A method for evaluating the therapeutic effect of a microRNA-controlled vaccinia virus according to  claim 1  on cancer in a cancer patient, comprising the steps of:
 (i) contacting the microRNA-controlled vaccinia virus with a cancer cell and a normal cell collected from the cancer patient; and 
 (ii) assaying the proliferation of the microRNA-controlled vaccinia virus in the cancer cell and the normal cell, wherein the microRNA-controlled vaccinia virus is determined to have therapeutic effect on cancer when proliferating in the cancer cell and not proliferating in the normal cell. 
 
     
     
         17 . The method according to  claim 16 , wherein the microRNA-controlled vaccinia virus has a fusion gene of a B5R gene and a marker gene, and the therapeutic effect on cancer is evaluated on the basis of marker expression.